Biological diagnostic techniques frequently require a purification process be performed on a sample prior to analysis. An unprocessed serum sample may contain as many as 105-106 different protein species at various concentrations. In proteomic diagnostics, accordingly, analytes of interest in a serum sample may be obscured by other protein species in the sample. Accordingly, microfluidic assay techniques typically require an off-chip purification technique such as dialysis, centrifugation, or desalting, prior to analysis. Requiring an off-chip purification step may limit the usefulness of microfluidic analysis techniques. Without the ability to receive and analyze a raw sample, the microfluidic device may not also serve as the collection point for a raw sample. Rather, the raw sample may first be processed in a macroscale device and later introduced into the microfluidic device for analysis.
Isoelectric fractionation is a technique for electrokinetically separating analytes in solution based on their isoelectric point. The isoelectric point of an analyte is the pH at which the analyte acquires no net charge. For example, proteins are composed of a variety of amino acid groups which act together to give the protein its overall charge. At the isoelectric point of the protein, the exchange of protons with the solution (protonation and deprotonation) will be balanced, and the protein acquires no net charge. At a pH below the isoelectric point of the protein, protonation typically dominates and the protein acquires a net positive charge. At a pH above the isoelectric point of the protein, deprotonation dominates and the protein acquires a net negative charge.
FIG. 1 depicts a tube 100 suitable for macroscale isoelectric fractionation.
The tube 100 is typically around a half-inch in diameter, and several inches long. The tube 100 includes individual compartments 105, 110, 115, and 120. Each chamber may be connected to the next by, for example, threaded connectors. Each compartment is separated from the next by a membrane cartridge 106, 111, 116, and 121, respectively. Each membrane cartridge 106, 111, 116, and 121 contains a porous membrane having a constant and specific pH value. The membrane cartridges and compartments may have O-rings or other sealing devices separating the individual compartments. The pH values of the membranes increase from a first end 130 of the tube 100 to a second end 135 of the tube 100. A sample in solution is loaded into the tube at any point and is separated using electrophoretic transport.
Electrophoretic transport involves applying an electric field across the tube 100. Accordingly, an electric field 140 is generated by applying a voltage across an anode 145 at the first end 130 of the tube and a cathode 150 at the second end 135 of the tube 100. Positively charged analytes will be transported through the tube 100 in the direction of the electric field 140, toward the cathode 150, and negatively charged analytes in the opposite direction. The analytes will pass through the membrane cartridges 106, 111, 116, 121 until they reach the compartment corresponding to their isoelectric point, at which point they will have no net charge, and will no longer move through the tube 100 by electrophoresis. In this manner, the analytes may be separated according to their isoelectric point. Following fractionation, the isolated samples in the compartments 105, 110, 115, and 120 may be removed for further analysis.
Macroscale isoelectric fractionation, as described above and with reference to FIG. 1, may take several hours to complete a fractionation. Furthermore, a large sample volume may be required to populate the macroscale compartments. The macroscale device may also be difficult to integrate with a microfluidic device. The membranes in the macroscale device are also polymerized in a chemical process with a support disk which is placed in the device following fabrication. The macroscale membrane must be mechanically robust to withstand the physical assembly steps. This limits the materials that may be used to form the macroscale membrane.